Development of complex tissues and organs requires specialized cells to differentiate from pluripotent progenitors. Initial cell fate specification ocurs early in embryogenesis. However, many adult tissues maintain a niche of progenitor cells that mediate repair upon damage. It is becoming increasingly clear that RNA-binding proteins play an important role in regulating the decision to differentiate. This is particularly true in early embryos, where nascent transcription has not yet begun, and in the brain, where the highly branched cellular morphology necessitates regulation at a distance from the nucleus. In this proposal, we describe a cohesive HTS assay development strategy to identify small molecule inhibitors of the RNA-binding proteins that regulate cell fate. The assays described include in vitro assays to identify molecules that block RNA-binding activity and cell-based assays to identify compounds that effect differentiation state. Once HTS-readiness and technical feasibility are established, these assays will be submitted to the NIH MLPCN for high throughput screening and probe identification. The compounds identified will be used to study the molecular mechanisms of cell fate specification in cell and animal models and in extracts. An independent outcome will be a direct comparison of the susceptibility of three different RNA- binding protein families to small molecules. This analysis may lead to development of more efficient target selection and library design strategy. Finally, the targets to be screened here are relevant to human diseases including brain cancer, multiple sclerosis, and parasitic infection. As such, it is possible that probes identified through HTS could eventually be developed into therapies. PUBLIC HEALTH RELEVANCE: This proposal describes plans to develop high throughput assays to screen for small molecules that inhibit RNA-binding proteins required to specify cell fate during embryogenesis and/or during tissue and organ repair. The strategy includes development of in vitro assays to screen for inhibitors, and cell or whole animal assays to study their effect on cell fate specification in vivo. Compounds identified will be used as research tool to dissect the contribution of post-transcriptional regulation to cellular differentiation. The assys could also identify pre-therapeutics that may eventually be developed to treat aggressive brain tumors such as glioblastoma multiforme, epithelial cancers such colon adenocarcinoma, dysmelinating disease including multiple sclerosis, inflammatory arthritis, and/or parasitic nematode infection. A third result of the proposed work will be a broad assessment of RNA-binding proteins as targets for small molecule intervention.